Gold Nanocluster-Decorated Nanocomposites with Enhanced

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Applications of Polymer, Composite, and Coating Materials

Gold Nanocluster-decorated Nanocomposites with Enhanced Emission and Reactive Oxygen Species Generation Junhan Xia, Xiaoyu Wang, Shuxian Zhu, Lu Liu, and Lidong Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19679 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 26, 2019

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ACS Applied Materials & Interfaces

Gold Nanocluster-decorated Nanocomposites with Enhanced Emission and Reactive Oxygen Species Generation Junhan Xia,#,† Xiaoyu Wang,#,*,† Shuxian Zhu,† Lu Liu,† and Lidong Li*,†,‡ †State

Key Laboratory for Advanced Metals and Materials, School of Materials

Science and Engineering, University of Science and Technology Beijing, Beijing 100083, P. R. China ‡State

Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian

116024, P. R. China

KEYWORDS: gold nanocluster, self-assemble, nanocomposite, enhanced emission, reactive oxygen species, cell death

ABSTRACT: Ligand-protected gold nanoclusters (AuNCs) show promise for high performance in biological applications, such as imaging and therapeutics. The assembly of AuNCs with biological macromolecules represents a simple but effective approach to fine tuning of material functionalities. Thus, these materials might enable intracellular applications of AuNCs. Herein, we prepared a new AuNC-based nanometric system through a self-assembly approach mediated by hydrophobic and electrostatic effects. We show that hydrophobic and electrostatic effects between 1

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fluorescent AuNCs with protamine and hyaluronic acid contribute to the formation of small nanocomposites with acceptable colloidal stability. More importantly, the AuNC-decorated nanocomposites show assembly-enhanced emission and singlet oxygen generation. In vitro experiments showed that our nanocomposites labeled specific cells by targeting CD44 and induced cell death by producing singlet oxygen. Hence, our AuNC-decorated nanocomposites show great potential as theranostic fluorescent nanomaterials.

1. Introduction Nanotechnology concerns the fabrication and application of functional materials at the nanoscale.1-3 Nanoparticles have unique optical and electronic properties that differ from those of their molecular and bulk regimes.4-6 Advances in nanotechnology have enabled the preparation of fluorescent nanoparticles for theranostic (i.e., therapeutic and diagnostic) biological applications. These materials have been developed into new tools for bioimaging, diagnostics, drug delivery, and disease therapy.7-10 Ideally, theranostic fluorescent nanoparticles should possess good dispersibility, stability and biocompatibility.11 Such fluorescent nanoparticles not only enable targeted cellular imaging but might also be used to treat various cancers.12 Recently, fluorescent nanoparticles, such as metal nanoclusters13,14, quantum dots (QDs),15,16 organic molecules,17,18 and composite nanoparticles19,20, have been prepared and investigated in biological applications. For noble metal nanoparticles, the motion of free electrons was limited by the size of nanoparticles. The collective 2


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oscillation of many electrons upon excited light produces surface plasmon resonance (SPR) effect.21 SPR effect can lead to photochemical generation of reactive oxygen species (ROS) and photothermal effects22-24, which can be used for photodynamic therapy. Unlike gold nanoparticles, metal nanoclusters formed from a few atoms are considered to be nanomaterials of interest with properties unlike those of metal nanoparticles.25-27 Discrete energy levels exist in metal nanoclusters, which give the metal nanoclusters molecular-like properties such as strong luminescence.28 Metal nanoclusters do not show SPR effect due to the obstructed collective oscillation of electrons.21 The existence of triplet excited states suggests potential for metal nanoclusters to function as photosensitizers.29 Energy transfer to oxygen molecules facilitates reactive oxygen species (ROS) generation,30 which is particularly effective for photodynamic therapy applications. These features suggest that metal nanoclusters have great potential in applications as theranostic fluorescent nanoparticles. However, the optical properties of metal nanoclusters are highly sensitive to the particle structure.31 The stability of ligand-protected metal nanoclusters used alone in complex biological environments is typically controlled by the stability of capping ligands on the surface of the nanoclusters.32 Thus, the structural integrity of metal nanoclusters can be compromised, leading to changes in their optical properties. To overcome these obstacles, metal nanoclusters can be used as ‘building blocks’ to design novel nanocomposites with controllable optical properties. Self-assembly is a powerful approach in nanotechnology for constructing novel nanomaterials from nanoparticle building blocks.33-35 The common configuration for 3



metal nanoparticles and polymer is the core–shell structure.36 Nanoparticles are the core and the organic molecules could be adsorbed or covalently bonded on the surface of nanoparticles to form a shell, which is a multi-step process.37,38 While for metal nanoclusters, surface ligands are required to stabilize their structures by strong metal– ligand interactions.39 The functional moieties of surface ligands, such as carboxyl groups and amino-groups,40 enable metal nanoclusters to assemble with biological molecules and carbohydrates. Thus, we can assemble metal nanoclusters with functional biomolecules to form biocompatible nanocomposites in a controlled way. In the self-assembly process, metal nanoclusters and biomolecules spatially organize into well-defined structures through non-covalent interactions. Without chemical modification, the unique optical properties of metal nanoclusters and biological characteristics of biomolecules are well maintained and combined in the nanocomposites. Notably, self-assembly approaches allow a higher concentration of metal nanoclusters to be aggregated in the nanoscale range.41 Metal nanocluster-based nanocomposites might also generate aggregation-induced emission,42 which further extends their applications in biological fields. In this paper, we aim to develop a new nanometric system based on gold nanoclusters (AuNCs) embedded in a polysaccharide/polypeptide hybrid material. We selected polysaccharides and polypeptides owing to their versatility and biocompatibility. Hyaluronic acid (HA), a negatively charged polysaccharide made of repeating disaccharide units, forms supramolecular aggregates with the strongly charged cationic protein protamine (PROT) via ionic crosslinking (Scheme 1).43 The 4


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introduction of glutathione (GSH) protected AuNCs induces both electrostatic attractions and hydrophobic effects enabling tight binding between HA and PROT; thus, stable nanocomposites are formed. The AuNCs are also densely loaded in the resulting nanocomposites, which have a unique combination of advantages, namely assembly-enhanced emission and singlet oxygen (1O2) generation. The specific binding ability of HA to CD44 antigens on cell surfaces44, enables these AuNC-based nanocomposites to target specific cells for imaging and the 1O2 generation adds to the theranostic capability. This self-assembly approach provides a new strategy for preparing theranostic fluorescent nanoparticles.

Scheme 1. Schematic illustration of the preparation and application for AuNC-HA-PROT nanocomposites with assembly-enhanced emission and ROS generation. 2. MATERIALS AND METHODS 5



Materials. The HA (12 kDa) was purchased from Shanghai Future Biochemical Reagent Co., Ltd. The 4ʹ,6ʹ-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) were purchased from Dakewe Biotech Co., Ltd. Anthracene-9,10-dipropionic acid disodium salt (ADPA) and p-Phthalic acid (PTA) were obtained from Aladdin reagent company. Fluorescein isothiocyanate (FITC) labeled anti-CD44 antibody was purchased from Abcam. Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H2O), GSH, 2ʹ,7ʹ-dichlorofluorescin diacetate and PROT, and all other chemicals were purchased from Sigma-Aldrich. The human breast cancer cells MDA-MB-231 and MCF-7 cells were obtained from the Chinese Academy of Medical Sciences. Preparation of AuNC-HA-PROT nanocomposites. GSH protected Au NCs were synthesized according to a previously reported method.45 Briefly, HAuCl4 (20 mM, 0.5 mL) and GSH (100 mM, 0.15 mL) were mixed with ultrapure water (4.35 mL). The resulting mixture was heated to 70 °C and maintained stirring (500 rpm) for 24 h. The resultant AuNC solution was dialyzed against ultrapure water. Then, the purified AuNC solution (400 μL) was diluted with ultrapure water (1.2 mL). A portion of NaOH solution (1 M, 2 μL) was then injected into the solution and 200 μL of HA and 200 μL of PROT solution were added to the mixture with the concentration of 1 mg/mL under stirring (500 rpm). In this way nanocomposites with different PROT concentrations were prepared. Hyaluronic acid: protamine ratio ranges from 1:4 to 4:1 (w/w). We also prepared HA-PROT nanocomposites without the AuNC as control group. Assay by isothermal titration microcalorimetry (ITC). The interactions of 6


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GSH-protected AuNC with HA were determined at 25 °C. We performed 20 injections of 2 μL HA solution (1 mg/mL) into 200 μL AuNC solution (0.2 mg/mL) in three repeat experiments. The dilution heat was determined by injecting HA solution into water through ITC. The titration data were fitted and analyzed according to our reported literature.46 We also used this method to determine the interactions of GSH-protected AuNC with PROT. ROS-detection of AuNC-HA-PROT nanocomposites. The ROS fluorescent probe 2ʹ,7ʹ-dichlorofluorescin (DCFH) was prepared according to a published procedure.47,48 2′,7′-dichlorofluorescin diacetate ethanol solution (500 μL, 1 mM) was added to NaOH solution (2 mL, 0.01 mM). The mixture was kept in the dark for 30 min for hydrolysis and diluted with 10 mL PBS to obtain DCFH solution. Then, 200 μL of the AuNC-HA-PROT nanocomposites, AuNC and HA-PROT nanocomposites were injected into 1 mL of DCFH solution (40 μM), respectively. The white light source (400−800 nm) was a xenon lamp (CXE-350, Beijing OPT Photoelectric Technology Co., Ltd). Each solution was irradiated under white light (1 mW/cm2) at 1 min intervals. The fluorescence spectra were measured with 488-nm excitation. Hydroxyl radicals (HO•) was determined by 3 mM PTA solution containing 2 mM NaOH. 200 μL of the AuNC-HA-PROT nanocomposites were injected into 1 mL of PTA solution. With white light (10 mW/cm2) irradiation at 5 min intervals, the fluorescence spectra were measured with 310-nm excitation. 1O2 was measured in ADPA solution (0.03 mg/mL in D2O). 200 μL of the AuNC-HA-PROT nanocomposites were injected into 2 mL of ADPA solution. With white light (10 7



mW/cm2) irradiation, the absorption spectra were measured at 5 min intervals. Cell culture and targeted imaging assay. The 100 000 MDA-MB-231 cells and MCF-7 cells were seeded in 35 mm culture dish, respectively. The culture medium is Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% (v/v) fetal calf serum. Then, the culture medium was replaced with 1 mL of culture medium containing 100 μL of the AuNC-HA-PROT nanocomposites. After incubation at 37 °C for 4 h, the medium was removed and the cells were washed with PBS (pH = 7.4). The specimens were observed with an oil immersion lens (100× magnification; NA 1.4) in an fluorescence microscope with a 420/40-nm excitation light source. As a control group, MDA-MB-231 cells were incubated with AuNC solution under the same conditions. A 3D fluorescence image was obtained by confocal laser scanning microscope through changing the z-focus. In order to assay the targeting ability of the AuNC-HA-PROT nanocomposites, 100 000 MDA-MB-231 cells and MCF-7 cells were seeded in 35 mm culture dish, respectively. The cells were incubated with 200 μl of PBS containing 20 μL of FITC labeled anti-CD44 for 30 min and then washed with PBS. The specimens were observed on the fluorescence microscope with a 480/30-nm excitation light source. Afterwards, the MDA-MB-231 cells were incubated with 1 mL of culture medium containing 100 μL of the AuNC-HA-PROT nanocomposites for 4h. The specimens were observed using fluorescence microscope with a 420/40-nm excitation light source. Cell death by 1O2. The MDA-MB-231 cells were seeded in four wells of two 8


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96-well plates (1×104 cells/well). After washing with PBS, the cells were incubated with AuNC-HA-PROT nanocomposites for 4 h, and then 4 wells of cells in one plate irradiated with white light at a power density of 80 mW/cm2 for 15 min. Correspondingly, 4 wells of cells in another plate was cultured in the dark for the same time. Subsequently, the cells were incubated with DAPI and PI in the dark at 37 °C for 15 min and immediately observed with the fluorescence microscopy under excitation with 375/28- and 480/40-nm light sources. To observe the boundary between the live and dead cells, the 100 000 MDA-MB-231 cells were seeded in 35 mm culture dish. After incubation with AuNC-HA-PROT nanocomposites for 4 h, half of the dish was wrapped in tin foil. The other half dish was irradiated with white light under same condition. Then, the cells were stained with DAPI and PI to obtained fluorescence images. Cell

Viability

Assay.

The

cell

viability

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

was

bromide

evaluated (MTT)

by assay.

Typically, MDA-MB-231 and MCF-7 cells were seeded in 96-well plates (1×104 cells/well). The cells were treated with various concentrations of AuNC-HA-PROT nanocomposites (0–100 μg/mL) for 4 h, and then irritated with white light at a power density of 80 mW/cm2 for 15 min. Another group was incubated in the dark for 24 h at 37 °C. The MTT (1 mg/mL in PBS, 100 μL) was injected into each well and allowed to stain for 4 h. The absorbance at 520 nm was measured on microplate reader (BioTek Synergy HT, USA). Characterization. The morphology of the AuNC-HA-PROT nanocomposites was 9



imaged with a transmission electron microscope (TEM, JEM-2010) operating at an accelerating voltage of 120 kV. An energy-dispersive spectrometer (EDS) analyzer attached to the high-resolution TEM (HRTEM) was used to analyze the composition. The average hydrodynamic diameters and zeta-potential were measured with a Malvern Nano ZS90 instrument. Thermogravimetric Analysis (TGA) was conducted on a TA Instruments SDT Q600 system. Calorimetric measurements were performed on a Malvern MicroCal ITC200. Absorption spectra were collected at room temperature with a Hitachi U3900 spectrophotometer. Fluorescence spectra and phosphorescence spectra at 77 K were measured with a Hitachi F-7000 spectrofluorometer. NanoLog infrared fluorescence spectrometer was used to detect emission of 1O2. The absolute fluorescence quantum yield (QY) was measured on NanologR FluoroLog-3-2-iHR320 spectrofluorometer. Fluorescence lifetimes were measured with a Delta Flex spectrofluorometer. Fluorescent signals of the cells were recorded on Olympus 1X73 fluorescence microscopy and Olympus FV1000-IX81 confocal laser scanning microscope. 3. RESULTS AND DISCUSSION Physicochemical characterization of AuNC-HA-PROT nanocomposites A series of AuNC-HA-PROT nanocomposites were prepared with different weights of AuNC, HA, and PROT. Physicochemical properties of the nanocomposites are listed in Table 1. Because of the carboxyl groups of GSH, the GSH protected AuNC had a negatively charged surface with a ζ potential of approximately −17.6 mV. Thus, the cationic PROT acted as an interpolyelectrolyte, which interacted with the 10


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negatively charged HA and AuNC in the nanocomposites preparation. When the amount of AuNC and HA were maintained constant and the amount of PROT was increased, the size of the AuNC-HA-PROT nanocomposites increased. However, increasing the amount of PROT to 0.4 mg led to forming aggregates. Unlike the AuNC-HA-PROT nanocomposites, no HA-PROT nanocomposites existed in the HA and PROT mixture when the amount of PROT was 0.025 or 0.4 mg. The HA-PROT nanocomposite formation depends mainly on electrostatic interactions between HA and PROT; thus, a lack or excess of PROT might prevent effective ionic crosslinking of HA and PROT. More importantly, these results indicate that electrostatic interactions are not the only effect controlling the AuNC-HA-PROT nanocomposite formation. Specifically, the AuNC-HA-PROT nanocomposites formed with PROT amounts in the range of 0.025–0.2 mg were smaller than 100 nm. For PROT weight in the range of 0.05–0.2 mg, the HA-PROT nanocomposites formed were larger than 100 nm. Thus, the presence of the AuNC promoted the formation of compact nanocomposites. We next compare the ζ-potential of the nanocomposites in the absence and presence of AuNC (Figure 1a). The AuNC-HA-PROT nanocomposites showed more negative ζ-potentials than those of the HA-PROT nanocomposites. These results confirmed the strong interactions among AuNC, HA and PROT led to the formation of more compact

and

smaller

nanocomposites.

Comparing

the

HA-PROT

and

AuNC-HA-PROT nanocomposites containing same amount of PROT, the ζ-potential changed largest from 5.8 to −38.4 mV when AuNC were added at a PROT weight of 11



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0.1 mg. The uniform AuNC-HA-PROT nanocomposites had a low PDI value (0.17) when the PROT weight was 0.1 mg. Taken together, we selected 0.1 mg of PROT for the following studies on the AuNC-HA-PROT nanocomposite formation. Table 1. Physicochemical properties of nanocomposites with different weights of AuNC, HA, and PROT (measured three times). Feeding weight AuNC (mg)

0.27

HA (mg)

0.1

Hydrodynamic diameter (nm)

PDI

ζ potential （mV）

0.025

49.35

0.343

-44.5

0.05

75.81

0.292

-43.0

0.1

71.69

0.170

-38.4

0.2

91.04

0.153

-28.5

PROT (mg)

0.4

0

0.1

Aggregates

0.025

0.639

0.318

-26.4

0.05

168.4

0.296

-20.0

0.1

397.8

0.174

5.8

0.2

490.8

0.162

4.6

0.4

Aggregates

The TEM images confirmed that the AuNC-HA-PROT nanocomposites retained spherical shapes with uniform size (Figure 1c). We exposed the region to the electron beam to destroy the biomolecules, AuNC could be observed in Figure 1d. The EDS spectrum for Figure 1d showed signals from S and Au, which we attributed to the presence of S and Au atoms in the GSH protected AuNC (Figure S1). The results clearly demonstrated that AuNC were successfully encapsulated into the AuNC-HA-PROT nanocomposites. The high-magnification image shows that the diameter of most AuNC was approximately 2.5 nm (Figure 1e). The structure of the AuNC in the composite was unchanged from that of the free AuNC (Figure 1f), which further confirmed that self-assembly had no effect on the AuNC structure. Moreover, 12


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the composition of AuNC-HA-PROT nanocomposites was analyzed by TGA. As shown in Figure S2, the AuNC-HA-PROT nanocomposites degraded lesser than HA-PROT nanocomposites, which could be ascribed to the presence of AuNC. The proportion of metallic gold is about 15%, and organic molecules account for 85%. However, without the AuNC, the HA-PROT nanocomposite showed a much larger size of approximately 390 nm (Figure 1g). We monitored the stability of the HA-PROT and AuNC-HA-PROT nanocomposites by analyzing the evolution of the particle size over time (Figure 1b). The AuNC-HA-PROT nanocomposites maintained their size for at least 96 h. Conversely, the HA-PROT nanocomposite without AuNC rapidly dissociated within 48 h. The colloidal stability of the AuNC-HA-PROT nanocomposites is consistent with the strong interactions among the AuNC, HA, and PROT.

13



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Figure 1. (a) ζ-potential measurements of AuNC-HA-PROT and HA-PROT nanocomposites; (b) Stability of AuNC-HA-PROT and HA-PROT nanocomposites; (c) TEM image of AuNC-HA-PROT nanocomposites; (d) HRTEM image of AuNC-HA-PROT

nanocomposite

after

electron

beam

exposure;

(e)

High

magnification image of the region indicated in Figure 1d; (f) HRTEM images of AuNCs; (g) TEM image of HA-PROT nanocomposites. To understand the interactions between the AuNC and HA and those between AuNC and PROT, we conducted ITC measurements. We obtained titration plots by titrating HA into AuNC solutions, which were fitted with a single-binding site model 14


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(Figure 2a). As a result, the enthalpy changes (∆H) and entropy changes (T∆S) were both positive, which could reflect the hydrophobic effect between AuNC and HA. Owing to the large positive value of T∆S, the interaction of HA and AuNC is an entropy-driven process. In fact, water molecules have an important effect on interactions among many biological molecules, such as polysaccharides, polypeptides, and proteins.49 The hydration sites of HA, such as pyranose rings and carboxylates, bind a large number of water molecules (Figure 2c).50 The carboxyl and amino groups in GSH-protected AuNC interact with the hydration layers of HA, which induce the hydrated water to form a more ordered structure. This effect causes a decrease in the hydration degree of HA and an increase in the number of excluded hydration water. Thus, interaction between HA and AuNC appears to be hydrophobic, and large entropy changes arise from the exclusion of water molecules. By fitting the curve in Figure 2b, we identified two processes in the interaction of AuNC and PROT. The ∆H and T∆S values in the first process are both positive, which indicates a hydrophobic effect. The negative ∆H and positive T∆S of the second process demonstrate the existence of electrostatic interactions. Considering the structure of PROT, as shown in Figure 2c, the hydrophobic amino acid side chains of PROT, which previously aggregated inside the protein, interacted with the amino acid residues of GSH in the AuNCs. The aggregates of PROT were buried and the amount of excluded hydrated water increased, which further confirms the hydrophobic effect. Furthermore, the amino groups of PROT interact with the carboxylate groups of GSH through electrostatic attraction. The water molecules do not influence this process to an 15



appreciable extent. Thus, the GSH was involved in the interactions between AuNC and HA, and also between AuNC and PROT. Both hydrophobic and electrostatic effects act upon the AuNC-HA-PROT nanocomposites formation, making the nanocomposites compact and stable.

Figure 2. ITC data for titration of AuNCs solution with (a) HA and (b) PROT. (c) Schematic illustration of the formation of AuNC-HA-PROT nanocomposites. Optical properties of AuNC-HA-PROT nanocomposites Surface ligands play an important role in the fluorescence of AuNCs51; hence, the strong interactions among GSH, HA, and PROT might induce unique optical properties in the AuNC-HA-PROT nanocomposites. UV−vis absorption spectra of the 16


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nanocomposites are shown in Figure S3. The AuNC-HA-PROT nanocomposites exhibited a distinct absorption peak at 400 nm, whereas HA-PROT had no absorption at this position. The absorption at 400 nm is consistent with the absorption band of GSH-protected AuNC in water. We measured the corresponding fluorescence emission spectra (Figure 3a). The AuNC-HA-PROT nanocomposites and free-AuNC exhibited the same fluorescence emission at 605 nm. The HA-PROT nanocomposites had no fluorescence. These results confirm that there was no change in the structure of the GSH-protected AuNC after formation of AuNC-HA-PROT nanocomposites. However, the fluorescence intensity of the AuNC-HA-PROT nanocomposites was approximately five times as high as that of the free AuNC. The absolute QY of AuNC-HA-PROT nanocomposites were measured to be 7.4%, while absolute QY of AuNC is 1.91%. The emission efficiency of the AuNC-HA-PROT nanocomposites is much higher than free AuNCs. To explore the fluorescence enhancement of the AuNC-HA-PROT nanocomposites, we measured the fluorescence lifetime (Figure 3b). The emission of the AuNC in nanocomposites decayed much slower than that of the free-AuNCs. The calculated average lifetimes were 5.57 μs for the AuNC-HA-PROT nanocomposite and 3.45 μs for AuNCs in water. In our system without any quenching interactions, the fluorescence lifetime τ is related to the radiative rate kr and nonradiative rate knr: τ = 1/(kr + knr). Because the structure of the GSH-protected AuNC does not change, kr of AuNC is constant. We attributed the increased fluorescence lifetime for AuNC-HA-PROT to 17



the decreased knr. The fluorescence of AuNC was largely affected by charge transfer from GSH to the AuNC core.31 Based on the previous results, the strong interactions among GSH, HA, and PROT stabilized the local Au–S environment. Hence, the nonradiative transition of AuNCs in the AuNC-HA-PROT nanocomposites weakened, which increased the fluorescence emission. The nonradiative transitions of the AuNC-HA-PROT nanocomposites weakened and the fluorescence emission increased; however, we also expected an increase in phosphorescence emission and ROS production. As shown in Scheme 2, because intersystem crossing efficiency is associated with the AuNC,52 the efficiency is constant. Hence, the weakened nonradiative transitions of the AuNC might enhance phosphorescence emission. We measured the phosphorescence spectra at 77 K. The phosphorescence emission of AuNC-HA-PROT was 1.96 times as high as that of the AuNC shown in Figure 3c. Thus, generation of ROS might occur through interactions between excited triplet states of the photosensitizers and oxygen. We used DCFH as a probe to detect ROS generation. Non-emissive DCFH is rapidly oxidized by ROS to become highly fluorescent 2',7'-dichlorofluorescein (DCF). By monitoring the DCF fluorescence signal we confirmed ROS generation. Upon continuous irradiation of a DCFH solution containing AuNC, the fluorescence intensity at 525 nm gradually increased and became much higher than that of the blank group (Figure 3d). While without irradiation, there is almost no DCF fluorescence signal for AuNC-HA-PROT nanocomposites (Figure S4). However, the emission increase of DCF for the AuNC-HA-PROT nanocomposites was more rapid than that of the free AuNC. The 18


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fluorescence intensity change for the HA-PROT nanocomposites was similar to that of the blank group. This result clearly confirms that ROS were generated from AuNC under

irradiation

and

that

ROS

generation-modification

occurred

in

the

AuNC-HA-PROT nanocomposites. These results are in line with the enhanced phosphorescence emission. Furthermore, ROS generation mechanism was determined by two probes, PTA and ADPA. In type I, the photosensitizer under irradiation can directly interact with solvent to produce radical ions, which further react with oxygen molecules (3O2) to generate ROS including HO•, superoxide radical and so on.53 After interacted with HO•, PTA can transform to hydroxyterephthalate with fluorescence emission at 425 nm.54 As shown in Figure S5a and S5b, upon continuous irradiation of a PTA solution containing AuNC-HA-PROT nanocomposites for 20 min, no fluorescence signal at 425 nm was observed. It is same with blank group, which indicated that there is no HO• generation from AuNC-HA-PROT nanocomposites based on type I mechanism. In type II, the triplet state photosensitizer interacts with triplet state 3O2 to produce 1O2 through energy transfer.53 After interacted with 1O2, ADPA transformed to endoperoxide with a decrease in ADPA absorbance.55 Compared to blank group, the absorbance of ADPA solution containing AuNC-HA-PROT nanocomposites gradually decreases by continuous irradiation for 20 min (Figure S5c and S5d). This suggests that the main ROS generated from AuNC-HA-PROT nanocomposites is 1O2 based on Type II mechanism. Compared with the AuNC-HA-PROT nanocomposites in the N2 purged solution, an emission of 1270 nm was observed in the presence of oxygen. As the emission is character of 19



1O 48,56, 2

the result directly proves the

1O

2

production by AuNC-HA-PROT

nanocomposites (Figure S6). According Type II mechanism57, the excited AuNC afforded AuNC in excited triplet state through intersystem crossing. Then, the AuNC in excited triplet state transferred energy to oxygen to produce 1O2.

Figure 3. (a) Fluorescence spectra of AuNC-HA-PROT, AuNC and HA-PROT under excitation at 380 nm. Inset figure shows fluorescence images of AuNC-HA-PROT and AuNC with 365 nm UV lamp irradiation. (b) Fluorescence intensity decay and (c) phosphorescence spectra at 77 K of AuNC-HA-PROT and AuNC under excitation at 380 nm. (d) Fluorescence intensity of DCF at 525 nm under white light irradiation (1 mW/cm2) in the presence of AuNC-HA-PROT, AuNC and HA-PROT. Excitation wavelength was 488 nm.

20


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Scheme 2. Schematic illustration for enhanced emission and ROS generation of AuNC-HA-PROT nanocomposites. 3.3 Cell targeted imaging and selective cell killing of AuNC-HA-PROT nanocomposites. Owing to the enhanced fluorescence signals and 1O2 production, we applied the AuNC-HA-PROT nanocomposites to targeted cell imaging and tumor cell killing. First, we evaluated the fluorescence stability of the nanocomposites in biological solutions. As shown in Figure S7, comparing with the AuNC-HA-PROT nanocomposites in water (control group), the fluorescence intensity of the AuNC-HA-PROT nanocomposites maintained their initial intensity in the PBS and DMEM. The intensities only decreased to 90% after 12 h. Thus, the AuNC-HA-PROT nanocomposites possess good stability, which is beneficial for their further biological applications. Considering the good targeting ability of HA for the CD44 antigen, we selected MDA-MB-231 cells, which highly express CD44, as the positive group and MCF-7 cells as the negative control group.58 After incubating both cells with the AuNC-HA-PROT nanocomposites for 4 h, we observed bright 21



fluorescence signals from the MDA-MB-231 cells. Besides, the fluorescence images have been recorded at different depth of the cells to obtain a volume image. The overlapped fluorescence image in Figure S8 indicates that the AuNC-HA-PROT nanocomposites were internalized by MDA-MB-231 cells. There was no fluorescence from the MCF-7 cells at the same incubation time (Figure 4). These results indicate the uptake of AuNC-HA-PROT nanocomposites by MDA-MB-231 cells. To clarify the uptake process, MDA-MB-231 and MCF-7 cells were incubated with FITC-labeled anti-CD44 antibody, respectively. As shown in Figure S9, an obvious green emission of FITC for MDA-MB-231 cells was observed, while no fluorescence signals for MCF-7 cell. It indicated that the CD44 was highly expressed on the surface of MDA-MB-231 cell. Then, after incubation with the anti-CD44, MDA-MB-231 cells and AuNC-HA-PROT nanocomposites were co-cultured for another 4 hours. As expected, the MDA-MB-231 cells showed a very weak fluorescence signal of AuNC-HA-PROT nanocomposites (Figure S10). It is a convincing proof that anti-CD44 block the entrance of AuNC-HA-PROT nanocomposites. The uptake of AuNC-HA-PROT nanocomposites by MDA-MB-231 cells is a CD44 receptor-mediated endocytosis process. Notably, the MDA-MB-231 cells showed weak fluorescence after incubation with free AuNC for 4 h (Figure S11), which confirmed that the assembly of HA and PROT promoted AuNC transport into MDA-MB-231 cells. Thus, the self-assembled AuNC-HA-PROT nanocomposites effectively harnessed the tumor cell killing performance of the AuNCs.

22


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Figure 4. Bright field, fluorescence and merged images of MDA-MB-231 and MCF-7 cells after incubation with AuNC-HA-PROT nanocomposites for 4 h. Scale bars: 5 μm. As shown above, we finally evaluated the tumor cell killing efficiency of the AuNC-HA-PROT nanocomposites in vitro. After incubating the MDA-MB-231 cells with the AuNC-HA-PROT nanocomposites for 4 h, we next irradiated these cells under white light at 80 mW/cm2 for 15 min. The viability of cells was determined by staining with DAPI and PI (Figure 5 and S12). DAPI stains both live and dead cells showing blue fluorescence, whereas PI only stains dead cells, showing red fluorescence. The cells showed mainly blue fluorescence (Figure 5a). The region subjected to white light irradiation, which showed red fluorescence (Figure 5b). The boundary between the live and dead cells is clearly shown in the overlay image (Figure 5c). However, for the blank group without AuNC-HA-PROT nanocomposites, the cells showed only blue fluorescence under white light irradiation or in the dark (Figure S13). It should be noted that, temperature can rise on gold nanoparticles under 23



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irradiation owing to the SPR effect.24 For AuNC, discrete energy levels obstructed the motion of electrons and then there is no SPR effect upon irradiation to produce heat 1O

(Figrue S14). Thus, these results indicate that

2

production from the

AuNC-HA-PROT nanocomposites caused cell death.

Figure 5. Fluorescence images of MDA-MB-231 cells after incubation with AuNC-HA-PROT nanocomposites stained with (a) DAPI, (b) PI and (c) their merged images. We conducted standard MTT to quantify the

1O

2-mediated

death of the

MDA-MB-231 cells. Without white light irradiation, more than 90% of the MDA-MB-231 cells remained alive after incubation with different AuNC-HA-PROT concentrations

(10–100

μg/mL)

(Figure

6a).

This

result

indicates

that

AuNC-HA-PROT nanocomposites showed nearly no cytotoxicity in the dark. However, as the AuNC-HA-PROT concentration increased to 100 μg/mL, the killing efficiency greatly improved and reached 83% under white light irradiation. Notably, the MDA-MB-231 cells were not killed by white light alone. After incubation of the MCF-7 cells with different AuNC-HA-PROT concentrations, the killing efficiencies were lower than 10% under both dark and light conditions (Figure 6b). These results confirmed that the AuNC-HA-PROT nanocomposite have excellent and selective 24


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killing activity toward MDA-MB-231 cells. This effect results from the light-induced toxic 1O2 produced by AuNC and the specific binding ability of HA to CD44 in the AuNC-HA-PROT nanocomposites.

Figure 6. Cell viability of (a) MDA-MB-231 cells and (b) MCF-7 after incubation with various concentrations of AuNC-HA-PROT nanocomposites under white light and in the dark. 4. CONCLUSION In this work, an AuNC-decorated stable nanocomposite with assembly-enhanced emission and ROS generation was generated through a self-assembly approach. The self-assembly process was mediated by hydrophobic and electrostatic effects among the AuNC, HA, and PROT. Highly stable AuNC-HA-PROT nanocomposites were obtained. The successful assembly of AuNCs in the nanocomposites produced remarkably enhanced emission and 1O2 generation. By targeting of CD44, the AuNC-HA-PROT nanocomposites were endocytosed to target MDA-MB-231 cancer cells for fluorescence imaging. We then confirmed the anti-cancer effects of the AuNC-HA-PROT nanocomposites in target MDA-MB-231 cells, indicating their great potential as a theranostic fluorescent nanomaterial. 25



ASSOCIATED CONTENT Supporting Information. EDS analysis and TGA of AuNC-HA-PROT nanocomposites. Ultraviolet-visible absorption of AuNC-HA-PROT, AuNCs and HA-PROT. ROS generation assay of AuNC-HA-PROT nanocomposites. Fluorescence ratios of AuNC-HA-PROT in different solutions. The overlapped fluorescence image of MDA-MB-231 cells after incubation with AuNC-HA-PROT. Fluorescence assay of MDA-MB-231 and MCF-7 cells after incubation with anti-CD44 and then with AuNC-HA-PROT. Fluorescence images of MDA-MB-231 after incubation with AuNCs. Fluorescence assays of MDA-MB-231 cells stained with DAPI and PI in different conditions. Temperature change of AuNC-HA-PROT nanocomposites upon irradiation. Acknowledgment We are thankful for the support from the National Natural Science Foundation of China (51673022, 51703009), the State Key Laboratory of Fine Chemicals (KF1613), the State Key Laboratory for Advanced Metals and Materials (2018Z-18) and the Fundamental Research Funds for the Central Universities (FRF-TP-16-026A1).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (X.W.). *E-mail: [email protected] (L.L.). 26


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ORCID Xiaoyu Wang: 0000-0002-2139-3152 Lidong Li: 0000-0003-0797-2518 Author Contributions #J.X.

and X.W. contributed equally to this study.

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Gold Nanocluster-Decorated Nanocomposites with Enhanced

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